Zn2+-chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors

ABSTRACT

Zn 2+ -chelating motif-tethered fatty acids as histone deacetylase (HDAC) inhibitors. Compounds performed well in in vitro and in vivo tests.

DESCRIPTION OF THE INVENTION

This invention was supported by Army Grant DAMD17-02-1-0117 and NationalInstitutes of Health Grant CA94829. The government has certain rights inthis invention. This application claims priority to U.S. Provisional60/526,348, filed Dec. 2, 2002.

FIELD OF THE INVENTION

The invention relates to histone deacetylase inhibitors, and inparticular, those including Zn²⁺-chelating motifs. More particularly,the invention relates to histone deacetylase inhibitors includingZn²⁺-chelating motifs, based on short-chain fatty acids.

BACKGROUND OF THE INVENTION

The acetylation status of core histones plays a pivotal role inregulating gene transcription through the modulation of nucleosomalpackaging of DNA (Kouzarides, “Histone acetylases and deacetylases incell proliferation.” Curr Opin Genet Dev 9: 40-48 (1999); Gray andEkstrom, “The human histone deacetylase family.” Exp Cell Res 262: 75-83(2001); Jenuwein and Allis, “Translating the histone code.” Science 293:1074-1080 (2001)). In a hypoacetylated state, nucleosomes are tightlycompacted, resulting in transcriptional repression due to restrictedaccess of transcriptional factors to their targeted DNA. Conversely,histone acetylation leads to relaxed nucleosomal structures, giving riseto a transcriptionally permissive chromatin state. A dynamic balancebetween the activities of histone acetyltransferases (HATs) and histonedeacetylases (HDACs), both of which are recruited to target genes incomplexes with sequence-specific transcription activators, maintainsthis level of this posttranslational modification. Aberrant regulationof this epigenetic marking system has been shown to cause inappropriategene expression, a key event in the pathogenesis of many forms of cancer(Wade, “Transcriptional control at regulatory checkpoints by histonedeacetylases: molecular connections between cancer and chromatin.” HumMol Genet 10: 693-698 (2001); Cress and Seto, “Histone deacetylases,transcriptional control, and cancer.” J Cell Physiol 184: 1-16 (2000);Marks et al., “Histone deacetylases and cancer: causes and therapies.”Nat Rev Cancer 1: 194-202 (2001)). Moreover, evidence demonstrates thatinhibition of HDAC triggers growth arrest, differentiation and/orapoptosis in many types of tumor cells by reactivating the transcriptionof a small number of genes (Jung, “Inhibitors of histone deacetylase asnew anticancer agents.” Curr Med Chem 8: 1505-1511 (2001); Grozinger andSchreiber, “Deacetylase enzymes: biological functions and the use ofsmall-molecule inhibitors.” Chem Biol 9: 3-16 (2002); Johnstone,“Histone-deacetylase inhibitors: novel drugs for the treatment ofcancer.” Nat Rev Drug Discov 1: 287-299 (2002); Kramer et al., “Histonedeacetylase as a therapeutic target.” Trends Endocrinol Metab 12:294-300 (2001); Marks et al., “Histone deacetylase inhibitors: inducersof differentiation or apoptosis of transformed cells.” J Natl CancerInst 92: 1210-1216 (2000)). Xenograft models also confirm these in vitrofindings, suggesting that modulation of HDAC's function is a target forthe prevention and/or therapeutic intervention of cancer.

To date, several structurally distinct classes of HDAC inhibitors havebeen reported (Jung, Curr Med Chem 8: 1505-1511 (2001); Grozinger andSchreiber, Chem Biol 9: 3-16 (2002); Johnstone, Nat Rev Drug Discov 1:287-299 (2002); Kramer et al., Trends Endocrinol Metab 12: 294-300(2001); Marks et al., J Natl Cancer Inst 92: 1210-1216 (2000)),including short-chain fatty acids (e.g., butyrate, valproate,phenylacetate, and phenylbutyrate) (Lea and Tulsyan, “Discordant effectsof butyrate analogues on erythroleukemia cell proliferation,differentiation and histone deacetylase.” Anticancer Res 15: 879-883(1995); Kruh, “Effects of sodium butyrate, a new pharmacological agent,on cells in culture.” Mol Cell Biochem 42: 65-82 (1982); Newmark andYoung, “Butyrate and phenylacetate as differentiating agents: practicalproblems and opportunities.” J Cell Biochem Suppl 22: 247-253 (1995);Phiel et al., “Histone deacetylase is a direct target of valproic acid,a potent anticonvulsant, mood stabilizer, and teratogen.” J Biol Chem276: 36734-36741 (2001)), benzamide derivatives (e.g., MS-27-275)(Suzuki et al., “Synthesis and histone deacetylase inhibitory activityof new benzamide derivatives.” J Med Chem 42: 3001-3003 (1999); Saito etal., “A synthetic inhibitor of histone deacetylase, MS-27-275, withmarked in vivo antitumor activity against human tumors. Proc Natl AcadSci U S A 96: 4592-4597 (1999)), trichostatin A (TSA) and analogues(Tsuji et al., “A new antifungal antibiotic, trichostatin.” J Antibiot(Tokyo) 29: 1-6 (1976); Jung et al. “Amide analogues of trichostatin Aas inhibitors of histone deacetylase and inducers of terminal celldifferentiation.” J Med Chem 42: 4669-4679 (1999); Furumai et al.“Potent histone deacetylase inhibitors built from trichostatin A andcyclic tetrapeptide antibiotics including trapoxin.” Proc Natl Acad SciU S A 98: 87-92 (2001)), hybrid polar compounds (e.g., suberoylanilidehydroxamic acid (SAHA)) (Richon et al., “A class of hybrid polarinducers of transformed cell differentiation inhibits histonedeacetylases.” Proc Natl Acad Sci U S A 95: 3003-3007 (1998);Remiszewski et al., “Inhibitors of human histone deacetylase: synthesisand enzyme and cellular activity of straight chain hydroxamates.” J MedChem 45: 753-757 (2002)), cyclic tetrapeptides (e.g., apicidin) (Kijimaet al., “Trapoxin, an antitumor cyclic tetrapeptide, is an irreversibleinhibitor of mammalian histone deacetylase.” J Biol Chem 268:22429-22435 (1993); Shute et al., “Analogues of the cytostatic andantimitogenic agents chlamydocin and HC-toxin: synthesis and biologicalactivity of chloromethyl ketone and diazomethyl ketone functionalizedcyclic tetrapeptides.” J Med Chem 30: 71-78 (1987); Han et al.,“Apicidin, a histone deacetylase inhibitor, inhibits proliferation oftumor cells via induction of p21WAF1/Cip1 and gelsolin.” Cancer Res 60:6068-6074 (2000); Nakajima et al., “FR901228, a potent antitumorantibiotic, is a novel histone deacetylase inhibitor.” Exp Cell Res 241:126-133 (1998)), and the depsipeptide FR901228 (Nakajima et al., ExpCell Res 241: 126-133 (1998)). Among these agents, short-chain fattyacids are the least potent inhibitors with IC₅₀ in the mM range, ascompared to that of μM or even nM for other types of HDAC inhibitors.Although the use of short-chain fatty acids in cancer treatment has beenreported, their therapeutic efficacy has been limited by the lowanti-proliferative activity, rapid metabolism, and non-specific mode ofaction.

Recently, X-ray crystallographic analysis of HDLP (histonedeacetylase-like protein), a bacterial HDAC homologue, has suggested adistinctive mode of protein-ligand interactions whereby TSA and SAHAmediate enzyme inhibition (Finnin et al., “Structures of a histonedeacetylase homologue bound to the TSA and SAHA inhibitors.” Nature 401:188-193 (1999)). The HDAC catalytic domain apparently consists of anarrow, tube-like pocket spanning the length equivalent to four tosix-carbon straight chains. A Zn²⁺ cation is positioned near the bottomof this enzyme pocket, which, in cooperation with two His-Aspcharge-relay systems, is believed to facilitate the deacetylationcatalysis.

Upon careful consideration of other work in the field, we realized thatthe weak potency of short-chain fatty acids in HDAC inhibition was, inpart, attributable to their inability to access the Zn²⁺ cation in theactive-site pocket, which we believe plays a pivotal role in thedeacetylation catalysis. Based on this realization and further study, westructurally modified short-chain fatty acids by tethering them with aZn²⁺-chelating motif via an aromatic linker. Our discoveries and studyhave led us to the invention of a new class of Zn²⁺-chelatingmotif-tethered short-chain fatty acids, some of which show inhibition ofHDAC activity and cancer cell proliferation in nM range, athree-orders-of-magnitude improvement over their parent compounds.

SUMMARY OF THE INVENTION

We realized that the weak potency of short-chain fatty acids as histonedeacetylase (HDAC) inhibitors was, in part, attributable to theirinability to access the zinc cation in the HDAC active-site pocket,which is believed to be important in deacetylation catalysis. Thepresent invention is based on structural modification of fatty acids,including the short-chain fatty acids, e.g., valproate, butyrate,phenylacetate, and phenylbutyrate. The present invention generallyincludes coupling fatty acids with Zn²⁺-chelating motifs (including, butnot limited to, hydroxamic acid and o-phenylene diamine) througharomatic ω-amino acid linkers. This strategy has led to the presentinvention, which includes a novel class of Zn²⁺-chelating motif-tetheredshort-chain fatty acids.

The efficacy of the inventive compounds in HDAC inhibition demonstratesthat potent HDAC inhibitors can be designed based on the frameworkprovided by the crystal structures of HDLP-ligand complexes. The presentinvention is based on a tethering strategy that allows the generation ofa large library of compounds via the divergent combination ofshort-chain fatty acids, ω-amino acids, and a zinc-chelator, such ashydroxamate.

The present invention includes histone deacetylase inhibitors having theformula:

wherein:

-   X is chosen from H and CH₃;-   Y is (CH₂)_(n) wherein n is 0-2;-   Z is chosen from (CH₂)_(m) wherein m is 0-3 and (CH)₂;-   A is a hydrocarbyl group;-   B is o-aminophenyl or hydroxyl group; and-   Q is a halogen, hydrogen, or methyl.

In one embodiment, A can comprise an aliphatic group, and the aliphaticgroup can be branched. A can also comprise an aromatic group, which maybe substituted or unsubstituted. In the formula, B can be o-aminophenylor hydroxyl. In some embodiments Y can be (CH2)_(n) wherein n is 0, Acomprises an aromatic group, B is hydroxy, and Q is hydrogen.

In some specific embodiments, m is 0 and X is H. Compounds having thesefeatures include, but are not limited to,

In some specific embodiments, X is H and A is chosen from:

wherein R comprises a branched or unbranched, substituted orunsubstituted, saturated or unsaturated, aliphatic or aromatic group.

The present invention also includes compositions comprising theinhibitor according to the invention, wherein the composition isenriched in the S-stereoisomer as compared to the R-stereoisomer.

Specific inhibitors of the invention includeN-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;N-Hydroxy-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;N-(2-Amino-phenyl)-4-(2-propyl-pentanoylamino)-benzamide;N-Hydroxy-4-(2-propyl-pentanoylamino)-benzamide; 2-Propyl-pentanoic acid{4-[2-amino-phenylcarbamoyl)-methyl]-phenyl}-amide; 2-Propyl-pentanoicacid (4-hydroxycarbamoyl-methyl-phenyl)-amide; 2-Propyl-pentanoic acid{4-[2-amino-phenylcarbamoyl)-ethyl]-phenyl}-amide; 2-Propyl-pentanoicacid [4-(2-hydroxycarbamoyl-ethyl)-phenyl]-amide; 2-Propyl-pentanoicacid {4-2-(2-amino-phenylcarbamoyl)-vinyl]-phenyl}-amide;2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-vinyl)-phenyl]-amide;N-(2-Amino-phenyl)-4-(butyrylamino-methyl)-benzamide;N-(2-Amino-phenyl)-4-(phenylacetylamino-methyl)-benzamide;N-(2-Amino-phenyl)-4-[(4-phenyl-butyrylamino-methyl]-benzamide;4-(Butyrylamino-methyl)-N-hydroxy-benzamide;N-hydroxy-4-(phenylacetylamino-methyl)-benzamide;N-hydroxy-4-[(4-phenyl-butyrylamino)-methyl]-benzamide;4-Butyrylamino-N-hydroxy-benzamide;N-hydroxy-4-phenylacetylamino-benzamide;N-hydroxy-4-(4-phenylbutyrylamino)-benzamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-butyramide;N-hydroxy-3-(4-phenylacetylamino-phenyl)-propionamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-4-phenyl-butyramide;N-(2-Amino-phenyl)-4-[(2-phenyl-butyrylamino-methyl]-benzamide;N-(2-Amino-phenyl)-4-[(3-phenyl-butyrylamino-methyl]-benzamide;N-hydroxy-4-(2-phenylbutyrylamino)-benzamide;N-hydroxy-4-(3-phenylbutyrylamino)-benzamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-phenyl-butyramide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-3-phenyl-butyramide;N-hydroxy-4-[(2-phenyl-butyrylamino)-methyl]-benzamide;N-hydroxy-4-[(3-phenyl-butyrylamino)-methyl]-benzamide;4-Benzoylamino-N-hydroxy-benzamide;4-(4-methyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-chloro)-Benzoylamino-N-hydroxy-benzamide;4-(4-bromo)-Benzoylamino-N-hydroxy-benzamide;4-(4-tert-butyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-phenyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-methoxyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-trifluoromethyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-nitro)-Benzoylamino-N-hydroxy-benzamide; Pyridine-2-carboxylic acid(4-hydroxycarbamoyl-phenyl)-amide;N-hydroxy-4-(2-methyl-2-phenyl-propionylamino)-benzamide;N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;N-hydroxy-4-(3-phenyl-propionylamino)-benzamide;4-(2,2-Dimethyl-4-phenyl-butyrylamino)-N-hydroxy-benzamide;N-hydroxy-4-[methyl-(4-phenyl-butyryl)-amino]-benzamide;N-hydroxy-4-(2-phenyl-propionylamino)-benzamide;N-hydroxy-4-(2-methoxy-2-phenyl-acetylamino)-benzamide;4-Diphenylacetylamino-N-hydroxy-benzamide;N-hydroxy-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;N-(2-Amino-phenyl)-4-phenylacetylamino-benzamide;N-(2-Amino-phenyl)-4-(5-phenyl-pentanoylamino)-benzamide;N-(2-Amino-phenyl)-4-(2-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(2,2-dimethyl-4-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-phenyl-propionylamino)-benzamide;N-(2-Amino-phenyl)-4-(4-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(2-methyl-2-phenyl-propionylamino)-benzamide;N-(2-Amino-phenyl)-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;and N-hydroxy-4-[2-(S)-phenylbutyrylamino]-benzamide;N-hydroxy-4-[2-(R)-phenyl butyrylamino]-benzamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(S)-phenyl-butyramide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(R)-phenyl-butyramide;N-hydroxy-4-(3-(S)-phenylbutyrylamino)-benzamide;N-hydroxy-4-(3-(R)-phenylbutyrylamino)-benzamide;N-hydroxy-4-[3-(S)-phenylbutyrylamino]-benzamide; andN-hydroxy-4-[3-(R)-phenylbutyrylamino]-benzamide.

The present invention also includes pharmaceutical compositionscomprising these inhibitors, and at least one pharmaceuticallyacceptable excipient. Still further, the invention includes methods ofinhibiting neoplastic cell proliferation in an animal, such as a human,comprising administering a therapeutically effective amount of at leastone inhibitor of the invention.

Additional features and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Thefeatures and advantages of the invention will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. The accompanyingdrawings, which are incorporated in and constitute a part of thisspecification, illustrate embodiments of the invention and together withthe description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows divergent conjugations of valproic acid with five aromaticω-amino acids and two Zn²⁺-chelating moieties to generate compounds1-10.

FIG. 2 shows HDAC inhibitory potency of compounds 1-10. In vitro HDACassay was carried out by using a commercial enzyme assay kit asdescribed herein. Data are the means±S.D. (n=3).

FIG. 3 shows structures and HDAC inhibitory potency of compounds 11-22.Values are the means±S.D. (n=3).

FIG. 4 shows the effect of HTPB, TSA, and phenylbutyrate on histoneacetylation and p21^(WAF/CIP1) expression in DU-145 cells. DU-145 cellswere exposed to HTPB, TSA, and phenylbutyrate at the indicatedconcentrations in 10% FBS-supplemented RPMI 1640 medium for 24 h. Anequivalent amount of protein from individual lysates was electrophoresedand probed by Western blot with respective antibodies. Actin was used asan internal reference protein.

FIG. 5 shows the growth inhibitory effect of HTPB on DU-145 cells. (A)Time course of the dose-dependent effect of HTPB on cell viability.DU-145 cells were treated with 0-2.5 μM HTPB in 10% FBS-containing RPMI1640 medium for the indicated times. Viable cells were examined by theMTT assay. Data are means±S.D. (n=6). (B) Dose-dependent effect of HTPBon the formation of nucleosomal DNA after 24-hr exposure. The formationof nucleosomes was quantitatively measured by Cell Death Detection ELISAwith lysates equivalent to 2×10³ cells for each assay. Data are theaverage of two independent determinations.

FIG. 6 illustrates examples of schemes for synthesizing compoundsaccording to the invention.

FIG. 7 (Frames 1-11) displays examples of different compounds accordingto the invention.

FIG. 8 (Frames 1 and 2) displays examples of zinc-chelating motifs thatcan be used in accordance with the invention.

FIG. 9 is a molecular modeling study of the ligand docking of compound19.

FIG. 10 diagrammatically illustrates the proton transfer that isbelieved to occur during the interaction of compound 19 and its bindingsite.

FIG. 11 is a molecular modeling study of the ligand docking of compound42.

FIG. 12 shows the effect of compound 42, SAHA, and TSA on histone H-4hyperacetylation and p21^(WAF/CIP1) expression in PC-3androgen-independent prostate cancer cells.

FIG. 13 shows the effect of compound 42, SAHA, and TSA on the activationstatus of Akt in PC-3 cells.

FIG. 14 shows the effect of compound 42 on several kinases.

FIG. 15 shows the effect of orally administered compound 42 on thegrowth of established PC-3 xenograft tumors.

FIG. 16 shows the effect of compound 42, compound 44, and SAHA on thegrowth of subcutaneous PC-3 xenograft tumors in athymic mice.

FIG. 17 shows Western blots of histone H3 and acetylated H3 in thehomogenates of two representative PC-3 tumors treated with compound 42,SAHA, or control.

FIG. 18 shows the dose-dependent antiproliferative effects of compound42 on representative breast, lung, and thyroid cancer cell lines.

FIG. 19 shows the effect of compound 42 on primary CLL cells.

FIG. 20 diagrammatically illustrates a chemical synthesis scheme forcompound 42.

FIG. 21 diagrammatically illustrates a generic chemical synthesis schemefor compounds according to the invention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In general, short-chain fatty acids exhibit HDAC inhibitory andantiproliferative activities in the mM range irrespective of thestructure of acyl chains (Grozinger and Schreiber, Chem Biol 9: 3-16(2002); Johnstone, Nat Rev Drug Discov 1: 287-299 (2002); Kramer et al.,Trends Endocrinol Metab 12: 294-300 (2001)). The present invention isbased on, among other things, the discovery that these fatty acids exertHDAC inhibition through non-specific hydrophobic interactions withsurface residues located at the enzyme pocket entrance and/or thehydrophobic region inside the tube-like pocket. We enhanced the HDACinhibitory potency of these short-chain fatty acids by tethering to aZn²⁺-chelating moiety through a hydrophobic spacer.

Measurement of the enzymatic activity of a histone deacetylase can beachieved using known methodologies. For example, Yoshida et al. (J.Biol. Chem. 265: 17174-17179 (1990)) describe the assessment of histonedeacetylase enzymatic activity by the detection of acetylated histonesin trichostatin A treated cells. Taunton et al. (Science 272: 408-411(1996)) similarly describes methods to measure histone deacetylaseenzymatic activity using endogenous and recombinant HDAC-1. Both Yoshidaet al. (J. Biol. Chem. 265:17174-17179, 1990) and Taunton et al.(Science 272: 408-411, 1996) are incorporated herein by reference.

Throughout this disclosure, reference will be made to compoundsaccording to the invention. Reference to such compounds, in thespecification and claims, includes esters and salts of such compounds.Thus, even if not explicitly recited, such esters and salts arecontemplated, and encompassed, by reference to the compounds themselves.

The fatty acids that can be used in accordance with the presentinvention comprise a hydrocarbyl portion and a carboxylic acid portion.As used herein, the term “hydrocarbyl” is understood to include“aliphatic,” “cycloaliphatic,” and “aromatic.” The hydrocarbyl groupsare understood to include alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, and alkaryl groups. Further, “hydrocarbyl” is understood toinclude both non-substituted hydrocarbyl groups, and substitutedhydrocarbyl groups, with the latter referring to the hydrocarbon portionbearing additional substituents, besides carbon and hydrogen.Additionally, while “carboxylic acid” is used to refer to the compounds,salts of such acids, i.e., carboxylates, are also expresslycontemplated. Moreover, carboxylic acids and carboxylates may be usedinterchangably herein.

In particular, fatty acids include, but are not limited to, those havingchain lengths comparable to an unbranched fatty acid of from about 3carbons to about 14 carbons in length. Thus, the chains can be, forexample, from about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbons inlength. The chains can be up to, for example, about 14, 13, 12, 11, 9,8, 7, 6, 5, or 4 carbons in length. The fatty acids can be straight orbranched and can include single, double, and/or triple bonds.Nonlimiting examples of fatty acids include valproate, butyrate,phenylacetate, and phenylbutyrate.

Zinc²⁺-chelating motifs contemplated in accordance with the presentinvention include, but are not limited to, hydroxamic acids ando-phenylene diamines. Other examples include trifluoromethyl ketone,α-keto amide, α-keto thiazole, 2-keto 1-methyl-1H-imidazole, α-keto1H-tetrazole, α-keto 1H-imidazole, 5-keto 1-methyl-1H-imidazole, α-ketooxazole, α-keto 4,5-dihydro-oxazole, α-keto benzooxazole, α-ketooxazolo[4,5-b]pyridine, and α-keto pyridine. Structures of these motifsare shown in FIG. 8.

The spacer can be any hydrocarbyl spacer, but preferably comprises anaromatic component. Aromatic linkers are believed to possess thefollowing advantages: 1) they enhance the structural rigidity of theconjugate, and 2) they increase van der Waals contacts with thetube-like hydrophobic region of the pocket to improve binding affinity.Examples of linkers include, but are not limited to, aromatic ω-aminoacids.

The linkers can exhibit lengths equivalent to that of four toeight-carbon straight chains, e.g., equivalent to 4, 5, 6, 7, or8-carbon straight chains. Thus, the lengths can be equivalent to 4-7 or4-6-carbon straight chains. The length may be based on the depth of thehydrophobic region of the binding pocket. Examples of linkers of theinvention include, but are not limited to, 4-(aminomethyl)benzoic acid,4-aminobenzoic acid, (4-aminophenyl)acetic acid,3-(4-aminophenyl)propionic acid, and 3-(4-aminophenyl)-acrylic acid.Among them, 4-(aminomethyl)benzoic acid has been used as the linker forMS-27-275 (Saito et al., Proc Natl Acad Sci U S A 96: 4592-4597 (1999)).

The following compounds are specifically contemplated:

-   N-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;-   N-Hydroxy-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;-   N-(2-Amino-phenyl)-4-(2-propyl-pentanoylamino)-benzamide;-   N-Hydroxy-4-(2-propyl-pentanoylamino)-benzamide;-   2-Propyl-pentanoic acid    {4-[2-amino-phenylcarbamoyl)-methyl]-phenyl}-amide;-   2-Propyl-pentanoic acid (4-hydroxycarbamoyl-methyl-phenyl)-amide;-   2-Propyl-pentanoic acid    {4-[2-amino-phenylcarbamoyl)-ethyl]-phenyl}-amide;-   2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-ethyl)-phenyl]-amide;-   2-Propyl-pentanoic acid    {4-2-(2-amino-phenylcarbamoyl)-vinyl]-phenyl}-amide;-   2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-vinyl)-phenyl]-amide;-   N-(2-Amino-phenyl)-4-(butyrylamino-methyl)-benzamide;-   N-(2-Amino-phenyl)-4-(phenylacetylamino-methyl)-benzamide;-   N-(2-Amino-phenyl)-4-[(4-phenyl-butyrylamino-methyl]-benzamide;-   4-(Butyrylamino-methyl)-N-hydroxy-benzamide;-   N-hydroxy-4-(phenylacetylamino-methyl)-benzamide;-   N-hydroxy-4-[(4-phenyl-butyrylamino)-methyl]-benzamide;-   4-Butyrylamino-N-hydroxy-benzamide;-   N-hydroxy-4-phenylacetylamino-benzamide;-   N-hydroxy-4-(4-phenylbutyrylamino)-benzamide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-butyramide;-   N-hydroxy-3-(4-phenylacetylamino-phenyl)-propionamide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-4-phenyl-butyramide;-   N-(2-Amino-phenyl)-4-[(2-phenyl-butyrylamino-methyl]-benzamide;-   N-(2-Amino-phenyl)-4-[(3-phenyl-butyrylamino-methyl]-benzamide;-   N-hydroxy-4-(2-phenylbutyrylamino)-benzamide;-   N-hydroxy-4-(3-phenylbutyrylamino)-benzamide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-phenyl-butyramide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-3-phenyl-butyramide;-   N-hydroxy-4-[(2-phenyl-butyrylamino)-methyl]-benzamide;-   N-hydroxy-4-[(3-phenyl-butyrylamino)-methyl]-benzamide;-   4-Benzoylamino-N-hydroxy-benzamide;-   4-(4-methyl)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-chloro)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-bromo)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-tert-butyl)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-phenyl)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-methoxyl)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-trifluoromethyl)-Benzoylamino-N-hydroxy-benzamide;-   4-(4-nitro)-Benzoylamino-N-hydroxy-benzamide;-   Pyridine-2-carboxylic acid (4-hydroxycarbamoyl-phenyl)-amide;-   N-hydroxy-4-(2-methyl-2-phenyl-propionylamino)-benzamide;-   N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;-   N-hydroxy-4-(3-phenyl-propionylamino)-benzamide;-   4-(2,2-Dimethyl-4-phenyl-butyrylamino)-N-hydroxy-benzamide;-   N-hydroxy-4-[methyl-(4-phenyl-butyryl)-amino]-benzamide;-   N-hydroxy-4-(2-phenyl-propionylamino)-benzamide;-   N-hydroxy-4-(2-methoxy-2-phenyl-acetylamino)-benzamide;-   4-Diphenylacetylamino-N-hydroxy-benzamide;-   N-hydroxy-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;-   N-(2-Amino-phenyl)-4-phenylacetylamino-benzamide;-   N-(2-Amino-phenyl)-4-(5-phenyl-pentanoylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(2-phenyl-butyrylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(2,2-dimethyl-4-phenyl-butyrylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(3-phenyl-propionylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(4-phenyl-butyrylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(3-phenyl-butyrylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;-   N-(2-Amino-phenyl)-4-(2-methyl-2-phenyl-propionylamino)-benzamide;-   N-(2-Amino-phenyl)-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;-   N-hydroxy-4-[2-(S)-phenylbutyrylamino]-benzamide;-   N-hydroxy-4-[2-(R)-phenylbutyrylamino]-benzamide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(S)-phenyl-butyramide;-   N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(R)-phenyl-butyramide;-   N-hydroxy-4-(3-(S)-phenylbutyrylamino)-benzamide;-   N-hydroxy-4-(3-(R)-phenylbutyrylamino)-benzamide;-   N-hydroxy-4-[3-(S)-phenylbutyrylamino]-benzamide; and-   N-hydroxy-4-[3-(R)-phenylbutyrylamino]-benzamide.

The compounds of the invention may be racemates, or racemic mixtures.The term “racemic” as used herein means a mixture of the (R)- and(S)-enantiomers, or stereoisomers, of the compounds of the invention, inwhich neither enantiomer, or stereoisomer, is substantially purifiedfrom the other.

The term “enriched,” as used herein to describe (R)- or(S)-stereoisomers of the invention, refers to a composition having agreater amount of the (R)-stereoisomer than (S)-stereoisomer, or viceversa. For example, the composition may contain greater than 50%, 55%,or at least about 60% of the (S)-stereoisomer of compound 42 by weight,based on the total weight of compound 42. In one embodiment, the amountof enriched (S)-compound 42 may be higher, for example, at least about65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any fraction thereof (i.e.,90.1%, 90.2%, etc.), of (S)-compound 42 by weight, based on the totalweight of compound 42. In a particular embodiment, the amount ofenriched (S)-compound 42 may be greater than 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or may be 100%, by weight,based on the total weight of compound 42. These terms also define theamount of any pharmaceutically acceptable salts of (S)-compound 42.These are non-limiting examples, and the same enrichments may beachieved for other racemic compounds of the invention.

The administration of enantiomerically enriched compositions of theinvention may result in a desirable therapeutic effect. That is,administration of enantiomerically enriched compositions may produce atherapeutic effect at a lower total concentration, or may reduce sideeffects resulting from the presence of the less-desirable enantiomer.These advantages are specifically contemplated.

Any of the inventive compounds, employed in the methods of theinvention, can be administered orally, parenterally (IV, IM, depot-IM,SQ, and depot-SQ), sublingually, intranasally (inhalation),intrathecally, topically, or rectally. Dosage forms known to those ofskill in the art are suitable for delivery of the inventive compoundsemployed in the methods of the invention.

Compositions are provided that contain therapeutically effective amountsof the inventive compounds employed in the methods of the invention. Thecompounds can be formulated into suitable pharmaceutical preparationssuch as tablets, capsules, or elixirs for oral administration or insterile solutions or suspensions for parenteral administration. Thecompounds described herein can be formulated into pharmaceuticalcompositions using techniques and procedures well known in the art.

About 0.1 to 1000 mg of an inventive compound or mixture of inventivecompounds employed in the methods of the invention, or a physiologicallyacceptable salt or ester is compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, flavor,etc., in a unit dosage form as called for by accepted pharmaceuticalpractice. The amount of active substance in those compositions orpreparations is such that a suitable dosage in the range indicated isobtained. The compositions can be formulated in a unit dosage form, eachdosage containing from about 1 to about 500 mg, or about 10 to about 100mg of the active ingredient. The term “unit dosage from” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

To prepare compositions, one or more inventive compounds employed in themethods of the invention are mixed with a suitable pharmaceuticallyacceptable carrier. Upon mixing or addition of the compound(s), theresulting mixture may be a solution, suspension, emulsion, or the like.Liposomal suspensions may also be used as pharmaceutically acceptablecarriers. These may be prepared according to methods known to thoseskilled in the art. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for lessening or ameliorating atleast one symptom of the disease, disorder, or condition treated and maybe empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers suitable for theparticular mode of administration. In addition, the active materials canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, or haveanother action. The compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

Where the compounds exhibit insufficient solubility, methods forsolubilizing may be used. Such methods are known and include, but arenot limited to, using co-solvents such as dimethylsulfoxide (DMSO),using surfactants such as TWEEN, and dissolution in aqueous sodiumbicarbonate. Derivatives of the compounds, such as salts or prodrugs,may also be used in formulating effective pharmaceutical compositions.

The concentration of the compound is effective for delivery of an amountupon administration that lessens or ameliorates at least one symptom ofthe disorder for which the compound is administered. Typically, thecompositions are formulated for single dosage administration.

The inventive compounds employed in the methods of the invention may beprepared with carriers that protect them against rapid elimination fromthe body, such as time-release formulations or coatings. Such carriersinclude controlled release formulations, such as, but not limited to,microencapsulated delivery systems. The active compound can be includedin the pharmaceutically acceptable carrier in an amount sufficient toexert a therapeutically useful effect in the absence of undesirable sideeffects on the patient treated. The therapeutically effectiveconcentration may be determined empirically by testing the compounds inknown in vitro and in vivo model systems for the treated disorder.

The compounds and compositions of the invention can be enclosed inmultiple or single dose containers. The enclosed compounds andcompositions can be provided in kits, for example, including componentparts that can be assembled for use. For example, an inventive compoundin lyophilized form and a suitable diluent may be provided as separatedcomponents for combination prior to use. A kit may include an inventivecompound and a second therapeutic agent for co-administration. Theinventive compound and second therapeutic agent may be provided asseparate component parts. A kit may include a plurality of containers,each container holding one or more unit dose of the inventive compoundemployed in the method of the invention. The containers can be adaptedfor the desired mode of administration, including, but not limited totablets, gel capsules, sustained-release capsules, and the like for oraladministration; depot products, pre-filled syringes, ampoules, vials,and the like for parenteral administration; and patches, medipads,creams, and the like for topical administration.

The concentration of active inventive compound in the drug compositionwill depend on absorption, inactivation, and excretion rates of theactive compound, the dosage schedule, and amount administered as well asother factors known to those of skill in the art.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound can be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules, or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, gum tragacanth, acacia, corn starch, orgelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a glidant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials, whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings, and flavors.

The active materials can also be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action. The inventive compounds can be used, for example, incombination with an antitumor agent, a hormone, a steroid, or aretinoid. The antitumor agent may be one of numerous chemotherapy agentssuch as an alkylating agent, an antimetabolite, a hormonal agent, anantibiotic, colchicine, a vinca alkaloid, L-asparaginase, procarbazine,hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide.Suitable agents include those agents which promote depolarization oftubulin. Examples include colchicine and vinca alkaloids, includingvinblastine and vincristine.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent such as water for injection, saline solution, fixed oil,a naturally occurring vegetable oil such as sesame oil, coconut oil,peanut oil, cottonseed oil, and the like, or a synthetic fatty vehiclesuch as ethyl oleate, and the like, polyethylene glycol, glycerin,propylene glycol, or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetates,citrates, and phosphates; and agents for the adjustment of tonicity suchas sodium chloride and dextrose. Parenteral preparations can be enclosedin ampoules, disposable syringes, or multiple dose vials made of glass,plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers include, but are notlimited to, physiological saline, phosphate buffered saline (PBS), andsolutions containing thickening and solubilizing agents such as glucose,polyethylene glycol, polypropyleneglycol, and mixtures thereof.Liposomal suspensions including tissue-targeted liposomes may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known in the art.

The inventive compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time-releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid, and the like.Methods for preparation of such formulations are known to those skilledin the art.

Compounds employed in the methods of the invention may be administeredenterally or parenterally. When administered orally, compounds employedin the methods of the invention can be administered in usual dosageforms for oral administration as is well known to those skilled in theart. These dosage forms include the usual solid unit dosage forms oftablets and capsules as well as liquid dosage forms such as solutions,suspensions, and elixirs. When the solid dosage forms are used, they canbe of the sustained release type so that the compounds employed in themethods of the invention need to be administered only once or twicedaily.

The oral dosage forms can be administered to the patient 1, 2, 3, or 4times daily. The inventive compounds employed in the methods of theinvention can be administered either three or fewer times, or even onceor twice daily. Hence, the inventive compounds employed in the methodsof the invention be administered in oral dosage form. Whatever oraldosage form is used, they can be designed so as to protect the compoundsemployed in the methods of the invention from the acidic environment ofthe stomach. Enteric coated tablets are well known to those skilled inthe art. In addition, capsules filled with small spheres each coated toprotect from the acidic stomach, are also well known to those skilled inthe art.

The inventive compounds employed in the methods of the invention mayalso be advantageously delivered in a nanocrystal dispersionformulations. Preparation of such formulations is described, forexample, in U.S. Pat. No. 5,145,684, the entire contents of which isincorporated by reference. Nanocrystalline dispersions of HIV proteaseinhibitors and their method of use are described in U.S. Pat. No.6,045,829, the entire contents of which is incorporated by reference.The nanocrystalline formulations typically afford greaterbioavailability of drug compounds.

The inventive compounds and methods can be used to inhibit neoplasticcell proliferation in an animal. The methods comprise administering toan animal having at least one neoplastic cell present in its body atherapeutically effective amount of at least one of the inventivecompounds, in compositions as described above. The animal can be amammal, including a domesticated mammal. The animal can be a human.

The term “neoplastic cell” is used to denote a cell that shows aberrantcell growth. The aberrant cell growth of a neoplastic cell includesincreased cell growth. A neoplastic cell may be, for example, ahyperplastic cell, a cell that shows a lack of contact inhibition ofgrowth in vitro, a benign tumor cell that is incapable of metastasis invivo, or a cancer cell that is capable of metastases in vivo and thatmay recur after attempted removal. The term “tumorigenesis” is used todenote the induction of cell proliferation that leads to the developmentof a neoplastic growth.

The terms “therapeutically effective amount” and “therapeuticallyeffective period of time” are used to denote treatments at dosages andfor periods of time effective to reduce neoplastic cell growth. As notedabove, such administration can be parenteral, oral, sublingual,transdermal, topical, intranasal, or intrarectal. When administeredsystemically, the therapeutic composition can be administered at asufficient dosage to attain a blood level of the inventive compounds offrom about 0.1 μM to about 100 mM. For localized administration, muchlower concentrations than this can be effective, and much higherconcentrations may be tolerated. One of skill in the art will appreciatethat such therapeutic effect resulting in a lower effectiveconcentration of the histone deacetylase inhibitor may vary considerablydepending on the tissue, organ, or the particular animal or patient tobe treated according to the invention. It is also understood that whilea patient may be started at one dose, that dose may be varied overtimeas the patient's condition changes.

The present invention provides compositions and methods for treating acell proliferative disease or condition in an animal, comprisingadministering to an animal in need of such treatment a therapeuticallyeffective amount of a histone deacetylase inhibitor of the invention. Asnoted, the animal can be a mammal, including a domesticated mammal. Theanimal can be a human.

The term “cell proliferative disease or condition” is meant to refer toany condition characterized by aberrant cell growth, preferablyabnormally increased cellular proliferation. Examples of such cellproliferative diseases or conditions include, but are not limited to,cancer, restenosis, and psoriasis. In some embodiments, the inventionprovides a method for inhibiting neoplastic cell proliferation in ananimal comprising administering to an animal having at least oneneoplastic cell present in its body a therapeutically effective amountof a histone deacetylase inhibitor of the invention. Cancers treatableaccording to the invention include, but are not limited to, prostatecancer, lung cancer, acute leukemia, multiple myeloma, bladdercarcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma,neuroblastoma, or melanoma.

It is contemplated that some compounds of the invention have inhibitoryactivity against a histone deacetylase from a protozoal source. Thus,the invention also provides a method for treating or preventing aprotozoal disease or infection, comprising administering to an animal inneed of such treatment a therapeutically effective amount of a histonedeacetylase inhibitor of the invention. Again, the animal can be amammal, and can be a human. In some embodiments, the histone deacetylaseinhibitor inhibits a protozoal histone deacetylase to a greater extentthan it inhibits mammalian histone deacetylases, particularly humanhistone deacetylases.

The present invention further provides a method for treating a fungaldisease or infection comprising administering to an animal in need ofsuch treatment a therapeutically effective amount of a histonedeacetylase inhibitor of the invention. The animal can be a mammal,including a human. The histone deacetylase inhibitor used according tothis embodiment of the invention can inhibit fungal histone deacetylaseto a greater extent than it inhibits mammalian histone deacetylases,particularly human histone deacetylases.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular compoundsemployed in the methods of the invention administered, the particularcondition being treated, the severity of the condition being treated,the age, weight, general physical condition of the particular patient,and other medication the individual may be taking as is well known toadministering physicians who are skilled in this art.

EXAMPLES Experimental

Chemical reagents and organic solvents were purchased from Aldrichunless otherwise noted. Nuclear magnetic resonance spectra (¹H NMR) weremeasured on Bruker 250 MHz. Chemical shifts (δ) are reported in partsper million (ppm) relative to TMS peak. Electrospray ionization (ESI)mass spectrometry analyses were performed with a 3-Tesla FinniganFTMS-2000 Fourier Transform mass spectrometer. Elemental analyses werewithin ±0.4% of calculated values.

Flash column chromatography was performed with silica gel (230-400mesh). The ω-amino acid methyl esters were prepared from thecommercially available acids using methanol/TMSCI, and(2-amino-phenyl)carbamic acid benzyl ester was synthesized fromo-phenylenediamine and benzyl chloride formate according to standardprocedures. Rabbit anti-acetyl-Histone H3 and H4 polyclonal antibodieswere purchased from Upstate Biotechnology (Lake Placid, N.Y.), Rabbitanti-p21 antibodies were from Santa Cruz Biotechnology (Santa Cruz,Calif.). Mouse monoclonal anti-actin was from ICN Biomedicals (Irvine,Calif.). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goatanti-mouse IgG were from Jackson ImmunoResearch (West Grove, Pa.).

Compounds 1-8 and 11-22 were synthesized according to methods a-edescribed as follows (Scheme 1A, FIG. 6), and compounds 9 and 10 wereprepared from 3-[4-(2-propyl-pentanoylamino)-phenyl]-acrylic acid bymethods f and g, respectively (Scheme 1B, FIG. 6), which are describedseparately under the title compounds.

Method a [1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride(EDC) coupling]. To a solution of individual short-chain fatty acids indry THF (5-10 mmol/mL) under N₂ was added various ω-amino acid methylester (1 equiv.), followed by EDC (1.3 equiv.). After stirringovernight, THF was removed under reduced pressure, and the residue wasdissolved in ethyl acetate (100 mL). The mixture was washedconsecutively with water (50 mL) twice and saturated brine (50 mL). Theorganic layer was dried over Na₂SO₄, and concentrated under vacuum. Theresulting residue was purified by silica gel flash chromatography.

Method b (ester cleavage). The resulting ester from Method a wasdissolved in a 2M KOH/MeOH solution. The mixture was stirred at 80° C.for 1 h, cooled to 0° C., acidified with 2N HCl to pH 3, concentratedunder vacuum, and ethyl acetate (100 mL) and H₂O (50 mL) were added. Theorganic phase was separated, washed consecutively with water andsaturated brine, 50 mL each, dried over Na₂SO₄, and concentrated undervacuum. The resulting residue was purified by silica gel flashchromatography.

Method c [bis(2-oxo-3-oxazolidinyl)phosphordiamidic chloride (BOP-Cl)coupling]. To a solution of the resulting acid from Method b in dry THF(5-10 mmol/mL) was added triethylamine (TEA, 1 equiv) under N₂. Themixture was stirred at room temperature for 10 min, and BOP-Cl (1.1equiv), O-benzylhydroxylamine hydrochloride (1 equiv), and TEA (3 equiv)were added. After stirring at room temperature overnight, the solutionwas concentrated under vacuum, and ethyl acetate (100 mL) was added,followed by 3% NaHCO₃ (50 mL). The organic phase was separated, andwashed consecutively with water and saturated brine, 50 mL each, driedover Na₂SO₄, and concentrated under vacuum. The resulting residue waspurified by silica gel flash chromatography.

Method d (EDC coupling). To a solution of individual acids resultingfrom Method b in dry THF (5-10 mmol/mL) under N2 was added(2-aminophenyl)carbamic acid benzyl ester (1 equiv), followed by EDC(1.3 equiv). After stirring overnight, the mixture was concentratedunder vacuum, and ethyl acetate (100 mL) was added. The organic phasewas washed consecutively with water (50 mL) twice, followed by saturatedbrine (50 mL), dried over Na₂SO₄, and concentrated. The resultingresidue was purified by silica gel flash chromatography.

Method e (hydrogenolysis). The N-benzyloxy or N-Cbz derivative,resulting from Method c or d, was dissolved in 1:1 methanol/THF (5-10mmol/mL), and 10% palladium on charcoal (10% w/w) was added. The mixturewas treated with hydrogen under atmospheric pressure for 2 h, andfiltered. The solvent was evaporated and the residue was recrystallizedwith ethyl acetate.

N-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide (1).¹H NMR (DMSO-d₆) δ 9.63 (s, 1H), 8.45 (t, J=6.1 Hz, 1H), 7.94 (d, J=7.8Hz, 2 H), 7.36 (d, J=8.3 Hz, 2H), 7.16 (d, J=7.6 Hz, 1H), 6.98 (t, J=7.5Hz, 1H), 6.78 (d, J=7.7 Hz, 1H), 6.60 (t, J=7.6 Hz, 1H), 4.90 (s, 2H),4.35 (d, J=5.9 Hz, 2H), 2.24 (m, 1H), 1.6-1.1 (m, 8H), 0.87 (t, 6H);HRMS: exact mass of (M+Na)⁺, 390.215195 amu; observed mass of (M+Na)⁺,390.21793 amu; anal. (C₂₂H₂₉N₃O₂) C, H, N.

N-Hydroxy-4-[(2-propyl-pentanoylamino)-methyl]-benzamide (2). ¹H NMR(DMSO-d₆) δ 11.17 (s, 1H), 9.00 (s, 1H), 8.40 (t, J=5.9 Hz, 1H), 7.70(d, J=8.2 Hz, 2H), 7.30 (d, J=8.2 Hz, 2H), 4.31 (d, J=5.7 Hz, 2H), 2.24(m, 1H), 1.6-1.1 (m, 8H), 0.85 (t, 6H); HRMS: exact mass of (M+Na)⁺,315.167911 amu; observed mass of (M+Na)⁺ 315.16755 amu; anal.(C₁₆H₂₄N₂O₃) C, H, N.

N-(2-Amino-phenyl)-4-(2-propyl-pentanoylamino)-benzamide (3). ¹H NMR(DMSO-d₆) δ 10.13 (s, 1H), 9.57 (s, 1H), 7.95 (d, J=8.7 Hz, 2H), 7.74(d, J=8.8 Hz, 2H), 7.16 (d, J=7.8 Hz, 1H), 6.98 (t, J=7.6 Hz, 1H), 6.78(d, J=8.0 Hz, 1H), 6.60 (t, J=7.7 Hz, 1H), 4.89 (s, 2H), 2.44 (m, 1H),1.7-1.1 (m, 8H), 0.89 (t, 6H); HRMS: exact mass of (M+Na)⁺, 376.199545amu; observed mass of (M+Na)⁺, 376.19762 amu; anal. (C₂₁H₂₇N₃O₂) C, H,N.

N-Hydroxy-4-(2-propyl-pentanoylamino)-benzamide (4). ¹H NMR (DMSO-d₆) δ11.07 (s, 1H), 10.13 (s, 1H), 8.94 (s, 1H), 7.65 (m, 4H), 2.42 (m, 1H),1.7-1.1 (m, 8H), 0.8 (t, 6H); HRMS: exact mass of (M+Na)⁺,301.152261amu; observed mass of (M+Na)⁺, 301.15194amu; anal.(C₁₅H₂₂N₂O₃) C, H, N.

2-Propyl-pentanoic acid{4-[(2-amino-phenylcarbamoyl)-methyl]-phenyl}-amide (5). ¹H NMR(DMSO-d₆) δ 9.84 (s, 1H), 9.33 (s, 1H), 7.56 (d, J=8.5 Hz, 2H), 7.26 (d,J=8.5 Hz, 2H), 7.14 (d, J=7.9 Hz, 1H), 6.90 (t, J=7.8 Hz, 1H), 6.72 (d,J=7.9 Hz, 1H), 6.53 (t, J=7.7 Hz, 1H), 4.83 (s, 2H), 2.41 (m, 1H),1.7-1.1 (m, 8H), 0.89 (t, 6H); HRMS: exact mass of (M+Na)⁺ 390.215195amu; observed mass of (M+Na)⁺, 390.21523 amu; anal. (C₂₂H₂₉N₃O₂) C, H,N.

2-Propyl-pentanoic acid (4-hydroxyphenylcarbamoylmethyl-phenyl)-amide(6). ¹H NMR (DMSO-d₆) δ 10.61 (s, 1H), 9.82 (s, 1H), 8.81 (s, 1H), 7.52(d, J=8.5 Hz, 2H), 7.16 (d, J=8.5 Hz, 2H), 3.22 (s, 2H), 2.38 (m, 1H),1.7-1.1 (m, 8H), 0.89 (t, 6H); HRMS: exact mass of (M+Na)⁺, 315.167911amu; observed mass of (M+Na)⁺, 315.16751 amu; anal. (C₁₆H₂₄N₂O₃) C, H,N.

2-Propyl-pentanoic acid{4-[2-(2-amino-phenylcarbamoyl)-ethyl]-phenyl}-amide (7). ¹H NMR(DMSO-d₆) δ 9.80 (s, 1H), 9.15 (s, 1H), 7.53 (d, J=8.4 Hz, 2H), 7.17 (d,J=8.4 Hz, 2H), 7.11 (d, J=7.8 Hz, 1H), 6.89 (t, J=7.9 Hz, 1H), 6.70 (d,J=7.9 Hz, 1H), 6.53 (t, J=7.7 Hz, 1H), 4.80 (s, 2H), 2.86 (t, J=7.9 Hz,2H), 2.59 (t, J=8.1 Hz, 2H), 2.38 (m, 1H), 1.7-1.1 (m, 8H), 0.87 (t,6H); HRMS: exact mass of (M+Na)⁺, 404.230845 amu; observed mass of(M+Na)⁺404.23043 amu; anal. (C₂₃H₃₁N₃O₂) C, H, N.

2-Propyl-pentanoic acid[4-(2-hydroxyphenylcarbamoyl)-ethyl)-phenyl]-amide (8). ¹H NMR (DMSO-d₆)δ 10.38 (s, 1H), 9.78 (s, 1H), 8.70 (s, 1H), 7.50 (d, J=8.5 Hz, 2H),7.10 (d, J=8.5 Hz, 2H), 2.75 (t, J=7.3 Hz, 2H), 2.40 (m, 1 H), 2.22 (t,J=7.4 Hz, 2H), 1.7-1.1 (m, 8H), 0.87 (t, 6H); HRMS: exact mass of(M+Na)⁺, 329.183561 amu; observed mass of (M+Na)⁺ 329.18295 amu; anal.(C₁₇H₂₆N₂O₃) C, H, N.

3-[4-(2-propyl-pentanoyl)-phenyl]-acrylic acid. This compound, aprecursor to compounds 9 and 10, was synthesized from 2-propyl-pentanoicacid (0.78 mL, 4.9 mmol) and 3-(4-amino-phenyl)-acrylic acid methylester (0.86 g, 4.9 mmol) according to Methods a and b aforementioned.Total yield, 1.05 g (70% for 2 steps); ¹H NMR (CDCl₃, 10% DMSO-d₆) δ9.49 (s, 1H), 7.71 (d, J=8.5 Hz, 2H), 7.55 (d, J=15.9 Hz, 1H), 7.45 (d,J=8.4 Hz, 2H), 6.31 (d, J=15.9 Hz, 1H), 2.40 (m, 1H), 1.7-1.1 (m, 8H),0.87 (t, 6H).

N-(2-Amino-phenyl)-3-[4-(2-propyl-pentanoylamino)-phenyl]-acrylamide(9). To a solution of 3-[4-(2-propyl-pentanoyl)-phenyl]-acrylic acid(200 mg, 0.7 mmol) in dry THF was added benzene-1,2-diamine (450 mg, 4.2mmol) under N₂, followed by EDC (180 mg, 0.9 mmol). After stirringovernight, the mixture was concentrated under vacuum, ethyl acetate (50mL) was added, and washed consecutively with water (30 mL) twice andsaturated brine (30 mL). The organic layer was dried over Na₂SO₄, andconcentrated under vacuum. The crude product was purified by flashchromatography (ethyl acetate-hexane, 1:1), giving compound 9 (200 mg,76% yield) as white solid. ¹H NMR (DMSO-d₆) δ 10.07 (s, 1H), 9.35 (s,1H), 7.71 (d, J=8.6 Hz, 2H), 7.56 (d, J=8.5 Hz, 2H), 7.51 (d, J=15.7 Hz,1H), 7.34 (d, J=6.6 Hz, 1H), 6.92 (t, J=7.1 Hz, 1H), 6.80 (d, J=15.5 Hz,1H), 6.75 (d, J=6.6 Hz, 1H), 6.58 (t, J=7.3 Hz, 1H), 4.96 (s, 2H), 2.44(m, 1H), 1.7-1.1 (m, 8H), 0.87 (t, 6H); HRMS: exact mass of (M+Na)⁺,402.215195 amu; observed mass of (M+Na)⁺, 402.21448 amu; anal.(C₂₃H₂₉N₃O₂) C, H, N.

N-Hydroxy-3-[4-(2-propyl-pentanoylamino)-phenyl]-acrylamide (10). To asolution of 3-[4-(2-propyl-pentanoylamino)-phenyl]-acrylic acid (100 mg,0.35 mmol) in dry DMF (3 mL) was added EDC (79 mg, 0.53 mmol) andhydroxybenzotriazole hydrate (HOBT) (62 mg, 0.46 mmol) under nitrogen.The mixture was stirred for 1 h, hydroxylamine hydrochloride (27.4 mg,0.39 mmol) and TEA (54 μL) were added, stirred for additional 12 h,concentrated under vacuum, and ethyl acetate (40 mL) and saturatedNaHCO₃ solution (15 mL) were added. The organic phase was separated, andwashed consecutively with water and saturated brine. 20 mL each. Theorganic layer was dried over Na₂SO₄, and concentrated under vacuum. Thecrude product was purified by flash chromatography [ethyl acetate-MeOH(9:1)], yielding compound 10 (45 mg, 40% yield) as white solid. ¹H NMR(DMSO-d₆) δ 10.69 (s, 1H), 10.07 (s, 1H), 9.00 (s, 1H), 7.67 (d, J=7.9Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.39 (d, J=15.6 Hz, 1H), 6.39 (d,J=15.3 Hz, 1H), 2.40 (m, 1H), 1.7-1.1 (m, 8H), 0.87 (t, 6H); HRMS: exactmass of (M+Na)⁺, 327.167911 amu; observed mass of (M+Na)⁺, 327.16809amu; anal. (C₁₇H₂₄N₂O₃) C, H, N.

N-(2-Amino-phenyl)-4-(butyrylamino-methyl)-benzamide (11). ¹H NMR(DMSO-d₆) δ 9.63 (s, 1H), 8.41 (t, J=5.7 Hz, 1H), 7.94 (d, J=8.1 Hz,2H), 7.36 (d, J=8.0 Hz, 2H), 7.17 (d, J=7.6 Hz, 1H), 6.98 (t, J=7.6 Hz,1H), 6.78 (d, J=8.1 Hz, 1H), 6.60 (t, J=7.7 Hz, 1H), 4.90 (s, 2H), 4.35(d, J=5.8 Hz, 2H), 2.15 (t, J=7.3 Hz, 2H), 1.60 (m, 2H), 0.88 (t, J=7.3Hz, 3H); HRMS: exact mass of (M+Na)⁺, 334.152595 amu; observed mass of(M+Na)⁺, 334.15221 amu; anal. (C₁₈H₂₁N₃O₂) C, H, N.

N-(2-Amino-phenyl)-4-(phenylacetylamino-methyl)-benzamide (12). ¹H NMR(DMSO-d₆) δ 9.60 (s, 1H), 8.66 (t, J=6.1 Hz, 1H), 7.92 (d, J=8.2 Hz, 2H), 7.31 (m, 7H), 7.16 (d, J=7.0 Hz, 1H), 6.98 (t, J=7.4 Hz, 1H), 6.78(d, J=6.6 Hz, 1H), 6.60 (t, J=7.4 Hz, 1H), 4.90 (s, 2H), 4.35 (d, J=5.7Hz, 2H), 3.51 (s, 2H); HRMS: exact mass of (M+Na)⁺, 382.152595 amu;observed mass of (M+Na)⁺, 382.15228 amu; anal. (C₂₂H₂₁N₃O₂) C, H, N.

N-(2-Amino-phenyl)-4-[(4-phenylbutyrylamino)-methyl]-benzamide (13). ¹HNMR (DMSO-d₆) 89.63 (s, 1H), 8.43 (t, J=6.1 Hz, 1H), 7.94 (d, J=8.3 Hz,2H), 7.34 (d, J=8.3 Hz, 2H), 7.21 (m, 6H), 6.98 (t, J=7.2 Hz, 1H), 6.78(d, J=8.11 Hz, 2H), 6.61 (t, J=6.5 Hz, 1H), 4.90 (s, 2H), 4.34 (d, J=6.1Hz, 2H), 2.59 (t, J=7.6 Hz, 2H), 2.20 (t, J=7.7 Hz, 2H), 1.84 (m, 2H);HRMS: exact mass of (M+Na)⁺, 410.183895 amu; observed mass of (M+Na)⁺,410.18232 amu; anal. (C₂₄H₂₅N₃O₂) C, H, N.

4-(Butyrylamino-methyl)-N-hydroxy-benzamide (14). ¹H NMR (DMSO-d₆) δ11.15 (s, 1H), 9.01 (s, 1H), 8.36 (t, J=5.6 Hz, 1H), 7.7 (d, J=8.1 Hz,2H), 7.32 (d, J=8.0 Hz, 2H), 4.30 (d, J=5.7 Hz, 2H), 2.15 (t, J=7.3 Hz,2H), 1.60 (m, 2H), 0.88 (t, J=7.3 Hz, 3H); HRMS: exact mass of (M+Na)⁺,259.10569 amu; observed mass of (M+Na)⁺, 259.10569 amu; anal.(C₁₂H₁₆N₂O₃) C, H, N.

N-Hydroxy-4-(phenylacetylamino-methyl)-benzamide (15). ¹H NMR (DMSO-d₆)δ 11.2 (s, 1H), 8.9 (s, 1H), 8.6 (t, J=5.8 Hz, 1H), 7.9 (d, J=8.3 Hz,2H), 7.28 (m, 7H), 4.04 (d, J=5.7 Hz, 2H), 3.5 (s, 2H); HRMS: exact massof (M+Na)⁺, 307.105311 amu; observed mass of (M+Na)⁺, 307.10512 amu;anal. (C₁₆H₁₆N₂O₃) C, H, N.

N-Hydroxy-4-[(4-phenylbutyrylamino)-methyl]-benzamide (16). ¹H NMR(DMSO-d₆) δ 11.2 (s, 1H), 9.0 (s, 1H), 8.4 (t, J=5.8 Hz, 1H), 7.7 (d,J=8.0 Hz, 2H), 7.21 (m, 7H), 4.3 (d, J=5.8 Hz, 2H), 2.58 (t, J=7.3 Hz,2H), 2.18 (t, J=7.3 Hz, 2H), 1.83 (m, 2H); HRMS: exact mass of (M+Na)⁺,335.136611amu; observed mass of (M+Na)⁺, 335.13716amu; anal.(C₁₈H₂₀N₂O₃) C, H, N.

4-Butyrylamino-N-hydroxy-benzamide (17). ¹H NMR (DMSO-d₆) δ 11.08 (s,1H), 10.09 (s, 1H), 8.94 (s, 1H), 7.67 (m, 4H), 2.31 (t, J=7.3 Hz, 2H),1.61 (m, 2H), 0.92 (t, J=7.4 Hz, 3H); HRMS: exact mass of(M+Na)⁺245.089661amu, Observed Mass of (M+Na)⁺245.08971amu, Difference<1.Oppm. Anal. (C₁₁H₁₄N₂O₃) C, H, N.

N-Hydroxy-4-phenylacetylamino-benzamide (18). ¹H NMR (DMSO-d₆) δ 11.1(s, 1H), 10.40 (s, 1H), 8.94 (s, 1H), 7.67 (m, 4H), 7.33 (m, 5H), 3.67(s, 2H); HRMS: exact mass of (M+Na)⁺, 293.089661 amu; observed mass of(M+Na)⁺, 293.08957amu; anal. (C₁₅H₁₄N₂O₃) C, H, N.

N-Hydroxy-4-(4-phenylbutyrylamino)-benzamide (19). ¹H NMR (DMSO-d₆) δ11.02 (s, 1H), 10.1 (s, 1H), 8.94 (s, 1H), 7.67 (m, 4H), 7.27 (m, 5H),2.63 (t, J=7.5 Hz, 2H), 2.35 (t, J=7.4 Hz, 2H), 1.87 (m, 2H); HRMS:exact mass of (M+Na)⁺, 321.120961amu; observed mass of (M+Na)⁺,321.11940amu; anal. (C₁₇H₁₈N₂O₃) C, H, N.

N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-butyramide (20). ¹H NMR(DMSO-d₆) δ 10.4 (s, 1H), 9.8 (s, 1H), 8.70 (s, 1H), 7.50 (d, J=8.4 Hz,2H), 7.10 (d, J=8.4 Hz, 2H), 2.75 (t, J=7.3 Hz, 2H), 2.24 (m, 4H), 1.61(m, 2H), 0.92 (t, J=7.4 Hz, 3H); HRMS: exact mass of (M+Na)⁺, 273.120961amu; observed mass of (M+Na)⁺, 273.12080 amu; anal. (C₁₃H₁₈N₂O₃) C, H,N.

N-Hydroxy-3-(4-phenylacetylamino-phenyl)-propionamide (21). ¹H NMR(DMSO-d₆) δ 10.36 (s, 1H), 10.14 (s, 1H), 8.70 (s, 1H), 7.50 (d, J=8.3Hz, 2H), 7.2-7.4 (m, 5H), 7.10 (d, J=8.3 Hz, 2H), 3.62 (s, 2H), 2.75 (t,J=7.5 Hz, 2H), 2.22 (t, J=7.6 Hz, 2H); HRMS: exact mass of (M+Na)⁺321.120961 amu; observed mass of (M+Na)⁺, 321.12040 amu; anal.(C₁₇H₁₈N₂O₃) C, H, N.

N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-4-phenyl-butyramide (22). ¹H NMR(DMSO-d₆) δ 10.36 (s, 1H), 9.80 (s, 1H), 8.70 (s, 1H), 7.52 (d, J=8.5Hz, 2H), 7.2-7.4 (m, 5H), 7.10 (d, J=8.4 Hz, 2H), 2.75 (t, J=7.4 Hz,2H), 2.62 (t, J=7.5 Hz, 2H), 2.15-2.4 (m, 4H), 1.8-2.0 (m, 2H); HRMS:exact mass of (M+Na)⁺, 349.152261 amu; observed mass of (M+Na)⁺,349.15223 amu; anal. (C₁₉H₂₂N₂O₃) C, H, N.

A list of all compounds, including additional compounds 23-67, is shownin FIG. 6.

In vitro HDAC assay. HDAC activity was analyzed by using a histonedeacetylase assay kit (Upstate Biotechnology, Lake Placid, N.Y.) byfollowing the manufacturer's instruction with slight modifications. Thisassay was based on the ability of DU-145 nuclear extract, which is richin histone deacetylase activity, to mediate the deacetylation ofbiotinylated [³H]-acetyl histone H4 peptide that was bound tostreptavidin agarose beads. The release of [³H]-acetate into thesupernatant was measured to calculate the HDAC activity. Sodium butyrate(0.25-1 mM) was used as a positive control.

Cell viability assay. The effect of HTPB on cell viability was assessedby the MTT {[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide]} assay in 96-well, flat-bottomed plates, in which 4,000 DU-145cells/well were seeded. Cells were exposed to HTPB at the indicatedconcentrations in 10% FBS-supplemented RPMI-1640 medium at 37° C. in 5%CO₂ for the indicated time. The medium was removed and replaced by 150μl of 0.5 mg/ml of MTT in RPMI-1640 medium, and cells were incubated inthe CO₂ incubator at 37° C. for 2 hours. Supernatants were removed fromthe wells, and the reduced MTT dye was solubilized with 200 μl/wellDMSO. Absorbance was determined on a plate reader at 570 nm. Eachtreatment was repeated in six wells.

Apoptosis Detection by An Enzyme-Linked Immunosorbent Assay (ELISA).Induction of apoptosis was assessed by using a Cell Death ELISA (RocheDiagnostics, Mannheim, Germany) by following the manufacturer'sinstruction. This test is based on the quantitative determination ofcytoplasmic histone-associated DNA fragments in the form ofmononucleosomes and oligonucleosomes after induced apoptotic death. Inbrief, 1×10⁶ DU-145 cells were cultured in a T-75 flask 24 h prior tothe experiment. Cells were treated with HTPB at the indicatedconcentrations in 10% FBS-supplemented RPMI 1640 medium. Both floatingand adherent cells were collected, cell lysates equivalent to 2×10³cells were used in the ELISA.

Western blot analysis. DU-145 cells (1×10⁶) treated with HTPB at theindicated concentrations in 10% FBS-supplemented RPMI 1640 medium for 24h were collected, and sonicated. Protein concentrations of the lysateswere determined by using a Bradford protein assay kit (Bio-Rad,Hercules, Calif.); equivalent amounts of proteins from each lysate wereresolved in 10% SDS-polyacrylamide gel, and then transferred ontoImmobilon-nitrocellulose membranes (Millipore, Bellerica, Mass.) in asemi-dry transfer cell. The transblotted membrane was washed twice withTris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). Afterblocking with TBST containing 5% nonfat milk for 40 min, the membranewas incubated with the primary antibody (1:1000 dilution) in TBST-1%nonfat milk at 4° C. overnight. After treatment with the primaryantibody, the membrane was washed three times with TBST for a total of15 min, followed by goat anti-rabbit or anti-mouse IgG-horseradishperoxidase conjugates (diluted 1:3000) for 1 h at room temperature andwash three times with TBST for a total of 1 h. The immunoblots werevisualized by enhanced chemiluminescence.

Results

For the first series of compounds, we employed valproic acid as lead tosynthesize Zn²⁺-tethered conjugates (FIG. 1) according to the proceduresdepicted in Scheme 1 (A, compounds 1-8; B, compounds 9 and 10). Forcompounds 1-8, valproic acid was coupled with four different ω-aminoacid methyl ester spacers via EDC activation. The resulting esters werecleaved to acids via alkaline hydrolysis. Under typical peptide couplingconditions (BOPCl or EDC), the resulting acids were treated withBn-protected hydroxylamine and Cbz-protected o-phenylenediamine to form,after hydrogenolysis, the respective anilides and hydroxamic acids(Scheme 1A). Compound 9 and 10 were synthesized by direct coupling of3-[4-(2-propyl-pentanoyl)-phenyl]-acrylic acid with o-phenylenediamineand hydroxylamine, respectively, under typical EDC coupling condition(Scheme 1B).

Zn²⁺-chelating motif-tethered valproate derivatives. The divergentconjugation of valproic acid with five different aromatic linkers andsubsequently two Zn²⁺-binding motifs yielded compounds 1-10. Thesetethered conjugates displayed varying degree of HDAC inhibitory potency(FIG. 2), with IC₅₀ values ranging from 5 μM (compound 8) to 80 μM(compound 5). This potency was an eighty- to five-fold improvement overthat of the parent molecule (IC₅₀, 0.4 mM). Removal of the valproylmoiety or the Zn²⁺-chelating motif from any of these conjugatescompletely abolished the HDAC inhibitory activity (data not shown),indicating the importance of the acyl function and the Zn²⁺-chelatingmotif in the protein-ligand interactions.

Among the ten tethered conjugates examined, compounds 1, 2, 4, and 8represented the optimal derivatives (IC₅₀, 5-8 μM), followed by 3, 9,and 10 (IC₅₀, 10-20 μM). Relatively, the hydroxamates (compounds 2, 4,6, 8, and 10) were generally more potent than their phenylenediaminecounterparts (compounds 1, 3, 5, 7, and 9). Moreover, the aromaticlinker exhibited a subtle effect on HDAC inhibitory activity. Among thefive aromatic ω-amino acids examined, (4-aminophenyl)acetate gave riseto conjugates with the least HDAC inhibitory potency (5 and 6), while4-(aminomethyl)benzoate appeared to be optimal. For further structuralmodifications, compounds 1, 2, 4, and 8 were used as leads since all ofthem exhibited IC₅₀<10 μM.

Structural modification. The finding that removal of the valproyl groupcompletely abrogated the inhibitory activity of the conjugateunderscored the importance of the acyl moiety in interacting with theactive-site pocket. We thus substituted the valproyl group in compounds1, 2, 4, and 8 with a butyryl, phenylacetyl, or phenylbutyryl to enhancethe stereoelectronic effect on HDAC inhibition (FIG. 3). All thesederivatives showed an improved potency in HDAC inhibition as compared tothe valproyl counterparts. Among various acyl functions examined, therelative potency was in the order ofphenylbutyryl>phenylacetyl>butyryl>valproyl when conjugated to the samespacer and Zn²⁺-chelating motif.

Of these twelve derivatives, compound 19 was especially noteworthy. Thishydroxamate-tethered phenylbutyrate (HTPB), i.e., compound 19, exhibitedIC₅₀ of 44 nM, a four-order-of-magnitude improvement overphenylbutyrate. This compound was used to examine its effect on HDACactivity in DU-145 prostate cancer cells.

Effect of HTPB on histone acetylation and p21^(WAF/CIP1) expression inDU-145 prostate cancer cells. Histone hyperacetylation and increasedexpression of the cyclin-dependent kinase inhibitor p21^(WAF/CIP1)represent two hallmark features in association with intracellular HDACinhibition (Marks et al., Nat Rev Cancer 1: 194-202 (2001)).Consequently, we examined the effect of HTPB vis-à-vis TSA andphenylbutyrate on HDAC activity in DU-145 prostate cancer cells bycharacterizing the status of histone acetylation and p21^(WAF/CIP1)expression.

DU-145 cells were exposed to HTPB at 0.5, 1, 2.5 μM, TSA at 0.25 and 0.5μM, or phenylbutyrate at 1, 2.5, and 5 mM in 10% FBS-supplemented RPMI1640 medium for 24 h. Western blot analysis of the cell lysatesindicates that treatment of these agents gave rise to elevated levels ofacetylated histones H3 and H4, and p21^(WAF/CIP1) (FIG. 4). The effectof 1 μM HTPB on these biomarkers approximated that of 0.25 μM TSA, or2.5 mM phenylbutyrate. DU-145 cells displayed small but significantamounts of intrinsic p21^(WAF/CIP1), and the level increasedsubstantially after exposure to HTPB as low as 0.5 μM. Together, thesedata confirmed that HTPB targeted HDAC activity in DU-145 cells.

Effect of HTPB on DU-145 cell viability. Effect of HTPB on cancer cellviability was assessed in DU-145 cells in 10% FBS-supplemented RPMI-1640medium. These cells displayed high degree of sensitivity to HTPB, withIC₅₀ in growth inhibition of approximately between 0.5 and 1 μM (FIG.5A). As evidenced by DNA fragmentation, HTPB sensitized DU-145 cells toapoptosis in a dose-dependent manner (FIG. 5B). As shown, extensiveapoptosis occurred at 24 h when the drug concentration exceeded 1 μM,indicating that this cytotoxic effect was, at least in part,attributable to the induction of apoptosis by HDAC inhibition. Othercell lines examined including AN3CA endometrial cancer cells, and SW-48and HCT-15 colorectal cancer cells. These cancer cells were alsosusceptible to the cytotoxic effect of HTPB with similar potency (datanot shown).

Discussion

Herein, we present the development of a novel class of HDAC inhibitors,in which short-chain fatty acids were tethered to a Zn²⁺-chelatingmoiety through hydrophobic linkage. Our strategy in the development ofthese compounds was built on a working model provided by the unique modeof HDAC inhibition by TSA and SAHA (Finnin et al., Nature 401: 188-193(1999)). Our novel tethering strategy led to the discovery of HTPB (orcompound 19), which displays HDAC inhibitory and antiproliferativeactivities at sub-μM concentrations that are in line with that reportedfor SAHA (Richon et al., Proc Natl Acad Sci U S A 95: 3003-3007 (1998)).

We obtained two lines of evidence that HTPB targeted HDAC activity inseveral cancer cell lines. Specifically, treatment of DU-145 prostatecancer cells with HTPB at as low as 0.5 μM caused the hyperacetylationof histones H-3 and H-4 in a dose-dependent manner. Likewise,p21^(WAF/CIP1) expression was substantially upregulated in response toHTPB. In contrast, the parent molecule phenylbutyrate required at least2.5 mM to achieve the same intracellular effects on histone acetylationand p21^(WAF/CIP1) expression.

HTPB is structurally distinct from existing HDAC inhibitors, in many ofwhich the cap groups consist of polar, planar structures. For example,the cap groups of TSA, SAHA, and MS-275 contain dimethylaminophenyl,phenylamino, and pyridin-3-yl-methoxycarbonyl groups, respectively.Thus, we have further concluded that the active-site pocket exhibits ahigh degree of flexibility in accommodating cap groups with differentstereoelectronic properties. Our data indicate that phenylbutyryl andphenylacetyl were more effective than aliphatic acyl moieties infacilitating the binding of the conjugates to the active-site pocket.This discrepancy might, in part, be due to differences in electrondensity and/or steric hindrance imposed by the branched side chain. Withregard to the aromatic linker, 4-aminobenzoate appeared to be optimal totether phenylbutyryl with hydroxamate, of which the length wassufficient to make contacts at both ends of the pocket.

Design and synthesis of the 2nd-generation HDACinhibitors—Structure-based optimization of Compound 19. Compound 19(HTPB) is structurally distinct from existing HDAC inhibitors, many ofwhich have cap groups consisting of polar, planar structures, e.g., TSA,dimethylaminophenyl; SAHA, phenylamino; and MS-27-275,pyridin-3-yl-methoxycarbonyl. This finding suggests that the HDACactive-site pocket exhibits a high degree of flexibility inaccommodating cap groups with different stereo-electronic properties.

To envisage the ligand binding, we carried out molecular docking ofcompound 19 (FIG. 9, in red) and TSA (FIG. 9, in yellow) into theactive-site pocket of HDLP following energy minimization to compare themode of recognition of individual ligands. As shown in FIG. 9, bothligands adopt similar configurations in binding to the pocket. Thearomatic linker of compound 19, 4-aminobenzoate, provides an optimallength to tether phenylbutyryl with hydroxamate, allowing both functionsto make contacts at both ends of the pocket. It appears that thehydroxamic acid function [C(O)NH—OH] attributes to thefour-orders-of-magnitude increase in the HDAC inhibitory potency ofcompound 19 over phenylbutyrate.

The modeling data in FIG. 9 suggest that the role of the hydroxamate inligand binding is twofold. First, it chelates the Zn2+ cation. Second,it facilitates proton transfer from NH—OH to Nτ of His-131 with the aidof the His131-Asp173 charge relay system (FIG. 10). (See Vanommeslaeghe,K., Van Alsenoy, C., De Proft, F., Martins, J. C., Tourwe, D., andGeerlings, P. Ab initio study of the binding of Trichostatin A (TSA) inthe active site of histone deacetylase like protein (HDLP). Org BiomolChem, 1: 2951-2957, 2003, for ligand binding studies of TSA.)

As a consequence, binding of hydroxamate HDAC inhibitors, either TSA orcompound 19, give rise to a transfer of negative charge from Asp tohydroxamate, resulting in a salt bridge formation between the negativelycharged hydroxamate and the positive charges on Zn²⁺ and the imidazolering (central panel, FIG. 10). Mechanistically, this salt bridgerepresents a major force contributing to the binding ofhydroxamate-based ligands to the active site pocket, and underlies thedifferential free energy change (ΔΔG^(‡)=−5.4 kcal/mol) required for the10⁴-fold increase in HDAC inhibitory potency in association with theconversion of phenylbutyrate to compound 19 (IC₅₀, 0.4 mM versus 44 nM).

The role of this salt bridge in binding affinity is further underscoredby a 10-fold drop in HDAC inhibitory potency when the hydroxamate moietyof compound 19 was replaced by a phenylenediamine function (compound55), i.e., IC₅₀, 0.044 versus 0.4 μM. In contrast to hydroxamate,binding of the phenylenediamine group to the active site does notinvolve a proton transfer to Nτ of His¹³¹. Instead, the electron-richdiamine only chelates with the Zn²⁺ cation without forming charge-chargeinteractions with His¹³¹. Consequently, the binding affinity with aphenylenediamine-based ligand, e.g., compound 55, is significantlydiminished as compared to the hydroxamate counterpart.

This molecular docking also provided useful guidance for the subsequentmodification of compound 19. As shown in FIG. 9, the cap groups of TSAand compound 19 are located near a groove surrounded by Tyr⁹¹, Glu⁹²,Gly¹⁴⁰, and Phe¹⁴¹. In principle, this groove could provide flexibilityin accommodating cap groups with varying degrees of bulkiness, and couldthus be exploited to enhance HDAC inhibitory potency.

Accordingly, we replaced the phenylbutyryl moiety of compound 19 withvarious α-branched aromatic fatty acyl groups, of which the rationalewas twofold. First, the amide linkage between the phenylbutyryl groupand the linker in compound 19 might be susceptible to proteolyticdigestion. Increasing the bulkiness of the acyl function might enhancethe metabolic stability by rendering the amide linkage more stericallyhindered. Second, increase in the size of the acyl function mightincrease the hydrophobic bonding with the aforementioned groove, therebyenhancing the binding affinity.

This strategy led to a number of HDAC inhibitors with greater potencythan compound 19 (i.e., HTPB). The structures and IC₅₀ values of some ofthe representative derivatives are summarized in Table 1.

TABLE 1 Representative phenylbutyrate-based HDAC inhibitors

Designation R IC₅₀ (nM) 19

44 42

25 44

32 61

38

Among more than 80 derivatives that were synthesized, compound 42represents the optimal agent with IC₅₀ comparable to that of TSA.Molecular modeling analysis indicates that compound 42 assumed aconfiguration that juxtaposed with that of TSA inside the active-sitepocket. (See FIG. 11.) In addition, the isopropyl moiety resided insidethe groove, and might interact with the nearby hydrophobic residues.Therefore, compound 42 was selected for further in vitro and in vivocharacterizations.

Compound 42 and TSA mediate antiproliferative effects at both epigeneticand cellular levels—Identification of novel cellular targets. Compound42, SAHA, and TSA were subject to examinations of their effects onp21^(WAF/CIP1) expression and histone H-4 hyperacetylation in PC-3androgen-independent prostate cancer cells. FIG. 12 demonstrates thatthe potency of compound 42 in the induction of these biomarkers iscomparable to that of TSA, and is about fivefold higher than that ofSAHA.

The effect of these agents on the activation status of Akt in PC-3 cells(PTEN^(−/−)) was also examined. Akt is constitutively activated in PC-3cells due to lack of functional PTEN, which contributes to the androgenindependency and chemotherapeutic resistance of these cells. It isnoteworthy that both compound 42 and TSA caused significant Aktdephosphorylation at as low as 1 μM in PC-3 cells (FIG. 13). Incontrast, no appreciable effect of SAHA on phospho-Akt was noted atcomparable concentrations, suggesting subtle differences in the mode ofaction between SAHA and compound 42/TSA.

The effect of compound 42 on phospho-Akt in PC-3 cells is noteworthy inlight of the clinical application of this HDAC inhibitor inhormone-refractory prostate cancer cells, most of which exhibit PTENmutations. This dephosphorylating effect, however, is kinase-specific.Among a series of other signaling kinases examined, the phosphorylationlevel of FAK (focal adhesion kinase) and ERKs was diminished in adose-dependent manner, while that of p38 or JNK remained unaffected(FIG. 14).

We hypothesize that this dephosphorylation is mediated through proteinphosphatase 1 (PPl1) which has been reported to form complexes with HDACisozymes to facilitate its nuclear localization. We propose thattreatment of PC-3 cells with compound 42 or TSA results in thedisruption of HDAC-PP1 complexes in the nucleus, which leads to there-localization of PP1 into the cytoplasm to mediate thedephosphorylation of target kinases. Alternatively, this effect could bedue to the acetylation of heat shock protein (HSP)-90, which results indiminished binding to Akt and its subsequent degradation (Fuino, L.,Bali, P., Wittmann, S., Donapaty, S., Guo, F., Yamaguchi, H., Wang, H.G., Atadja, P., and Bhalla, K. Histone deacetylase inhibitor LAQ824down-regulates Her-2 and sensitizes human breast cancer cells totrastuzumab, taxotere, gemcitabine, and epothilone B. Mol Cancer Ther,2: 971-984, 2003).

In vivo characterization of the antitumor effects of compound 42 onprostate cancer. The effect of orally administered compound 42 (50 and100 mg/kg/day) vis-à-vis intraperitoneally injected SAHA (50 mg/kg/day)on the growth of established PC-3 xenograft tumors was examined. Inaddition, we also included compound 44 (50 and 100 mg/kg/day) in lightof its comparable HDAC inhibitory potency to that of compound 42 (IC₅₀,32 versus 25 nM). Both compounds 42 and 44 were effective in suppressingin vitro PC-3 cell proliferation in 10% FBS-containing medium in adose-dependent manner, with IC₅₀ values of 0.6 and 0.8 μM, respectively(FIG. 15, 72-h treatment).

FIG. 16 depicts the growth of subcutaneous PC-3 xenograft tumors inathymic mice treated with HDAC inhibitors as described above (Panel A,data presented as mean tumor volume±SD; Panel B, photographs ofrepresentative mice from groups treated with vehicle, compound 44 andcompound 42). As shown, both compounds 42 and 44 at either dose wereeffective in suppressing the growth of established PC-3 tumors via theoral route. The in vivo efficacy of oral compound 42 at 50 mg/kg/day wascomparable to that of i.p. SAHA at the same dose, which causedreductions of 70.4% and 69.9%, respectively, in the growth of the tumorsin comparison to the vehicle-treated control groups. At 100 mg/kg/day,compound 42 caused a nearly complete inhibition of PC-3 tumor growth(91.7% reduction) in the absence of overt toxicity as assessed bymonitoring body weights and pathological examination.

At the conclusion of the study, complete pathological evaluation of onemouse from each treatment group was performed by board-certifiedveterinary pathologists at The Ohio State University College ofVeterinary Medicine, which included clinical pathology (hematology,serum chemistries) and complete necropsy (gross and microscopicexamination of at least 27 different tissues and organs). Grosspathologic abnormalities observed at necropsy were limited to smalladhesions in the peritoneum of the SAHA-treated and DMSO-treated controlmice, which were likely secondary to the daily i.p. injections.Hematological parameters were within normal limits except for aneutrophilia, which was observed only in mice from both vehicle-treatedcontrol groups and the SAHA- and compound 44 (50 mg/kg)-treated groups.Serum chemistry values were consistent with mild dehydration in thevehicle-treated mice, but were within normal limits for all other mice.

To correlate biological response with the proposed mechanism of actionidentified in vitro, the ability of orally administered compound 42 andi.p. SAHA to modulate the acetylation of histone H-3 in PC-3 xenografttumors was assessed by immunoblotting. FIG. 17 depicts Western blots ofhistone H3 and acetylated H3 in the homogenates of two representativePC-3 tumors with different volumes from tumor-bearing mice treated withvehicle, oral compound 42 at 50 or 100 mg/kg/day, or i.p. SAHA at 50mg/kg/day for 28 days. A significant increase in the acetylation levelof histone H3 was noted in drug-treated groups, characteristic of invivo HDAC inhibition.

In the in vivo experiment, PC-3 cells, suspended in equal volumes ofserum-free medium and Matrigel basement membrane matrix, were injectedsubcutaneously into the flanks of 5-7 week old male NCr athymic nudemice (nu/nu) (0.5×10⁶ cells/0.1 ml/mouse). Daily treatment with compound42 or 44 (50 and 100 mg/kg/day) by oral gavage or SAHA (50 mg/kg/day) byintraperitoneal injection (N=6 for each group) began when tumor volumesreached 170-200 mm³. The control group received p.o. or i.p. vehicleonly (0.1% methylcellulose/0.05% Tween 80 in water and DMSO,respectively). Treatments continued until the mean tumor volume of thecontrol group reached approximately 1,500 mm³. Tumors were measuredevery week using Vernier calipers and their volumes calculated using astandard formula: width²×length×0.52. Body weights were measured weeklyand were stable throughout the study.

In vitro antiproliferative effects of compound 42 in breast cancer celllines, lung cancer cell lines, and thyroid cancer cell lines. Accordingto the sixty-cell line screening by the NCI Developmental TherapeuticsProgram (DPT), compound 42 exhibited potent in vitro antiproliferativeactivities with an average GI50 (50% inhibition of cell growth) value of0.2 μM against all 60 cell lines. In addition, we have tested compound42 in thyroid cancer cell lines. The dose-dependent antiproliferativeeffects of compound 42 in representative breast, lung, and thyroidcancer cell lines are shown in FIG. 18.

In vitro and in vivo antiproliferative effect of compound 42 in chroniclymphocytic leukemia (CLL). Compound 42 was tested in primary CLL cellsfor its ability to inhibit HDACs and promote cytotoxicity. As shown inFIG. 19, at the 1 μM and above concentration where HDACs are inhibited,significant cytotoxicity was observed (N=6). In addition, a pilot invivo study using the TLC-1 transgenic mouse model indicates a trendtoward improved survival and no notable toxicity with 100 mg/kg compound42 by oral gavage for 5 days on/two days off for 4 weeks.

Stereoselectivity

It was surprisingly discovered that the HDAC-inhibitory activity ofcompound 42 is stereoselective, of which the (S)-isomer is more potentthan the (R)-counterpart (IC50, 15 nM versus 80 nM).

In summary, by combining molecular modeling and combinatorial chemistrytechniques, short-chain fatty acids were used as scaffolds to develop anovel class of potent HDAC inhibitors. The optimal agent, compound 42(NSC-D-731438) inhibits HDAC activity with an IC₅₀ of 25 nM, a more than10,000-fold increase over that of its parent compound phenylbutyrate.Compound 42 exhibits several unique features that make it a promisingcandidate to be brought into the clinic. First, compound 42 mediates invitro antiproliferative effects through both epigenetic and cellularmechanisms, a feature that is similar to that of TSA, but is lacking inSAHA. Second, it is orally bioavailable with in vitro and/or in vivopotency superior to than that of MS-275 and SAHA. Third, it has nodemonstrable toxicity in tumor-bearing mice after a 28-day course at thedose of 50 or 100 mg/kg/day, which offers the opportunity for chronicadministration and combination with other targeted therapies. Finally,compound 42 has a simple structure, and is amenable to large-scalesynthesis.

Production of Compound 42

The inventors have synthesized compound 42 numerous times in multi-gramscales. Basically, it has been prepared via a four-step synthesis withoverall yield of 52% (FIG. 20). All starting materials are readilyavailable, and the synthesis is amenable to scale-up to multi-hundredgram levels in a laboratory setting, of which the procedures aredescribed as follows. The intermediates and final product could beisolated by crystallization from the reaction mixture with puritygreater than 99% without using chromatographic separation. Overall, thesynthesis and purification procedures are straightforward, and there isno special concern regarding the large-scale production of thiscompound.

N-(4-Acetyl-phenyl-3-methyl-2-phenyl-butyramide (1).(α-Isopropyl)-phenylacetic acid (4 g, 22.3 mmol) was dissolved inthionyl chloride (30 ml), and heated at 50° C. for 1 hr. After removingthe solvent, the residue was dissolved in THF (50 ml), andp-aminobenzoic acid methyl ester (3.4 g, 22.5 mmol) and Et₃N (3.5 ml, 25mmol) in THF (50 ml) were added with stirring at room temperature. After4 hr, THF was removed under vacuum, dissolved in ethyl acetate (200 ml),and washed, in tandem, with water (100 ml) and brine (100 ml). Theorganic layer was dried over Na₂SO₄, and concentrated under vacuum,yielding compound 1 (5.6 g; yield 85%). The crude product was used forthe next step without further purification.

4-(3-methyl-2-phenyl-butyrylamino)benzoic acid (2). Compound 1 (5.6 g;20 mmol) was dissolved in methanol (150 ml) containing KOH (21 g). Themixture was refluxed for 2 hr, cooled to 0° C., and 30 ml of 12 N HCl(12N) was added dropwise to precipitate out the product. Solvent wasremoved under vacuum, and cold water (120 ml) was added. The precipitatewas collected by filtration, washed with water, and dried, yieldingcompound 2 (5 g; yield 84%).

N-Benzyloxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (3). To asolution of 2 (5 g; 16.8 mmol) in dry THF (120 ml) was addedtriethylamine (TEA, 2.5 ml; 16.8 mmol) under N₂. The mixture was stirredat room temperature for 10 min, andbis(2-oxo-3-oxazolidinyl)phosphordiamidic chloride (BOP-Cl) (4.7 g; 18.7mmol), O-benzylhydroxylamine hydrochloride (2.7 g; 17 mmol), and TEA(7.5 ml) were added. After stirring at room temperature overnight, thesolution was concentrated under vacuum, and ethyl acetate (200 ml) wasadded, followed by 3% NaHCO₃ (80 ml). The organic phase was separated,and washed consecutively with water and saturated brine, 100 ml each,dried over Na₂SO₄, and concentrated under vacuum. The residue (6.1 g,90% yield) was used directly for hydrogenolysis without furtherpurification.

N-Hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (4). Compound 3(6.1 g; 15.2 mmol) was dissolved in 1:1 methanol/THF (120 ml), and 10%palladium on charcoal (0.6 g, 10% w/w) was added. The mixture wastreated with hydrogen under atmospheric pressure for 2 h, and filtered.The solvent was evaporated and the residue was recrystallized with ethylacetate, yielding 4.2 g (90% yield).

An alternative scheme for synthesis of HDAC inhibitors according to thepresent invention is illustrated in FIG. 21.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A histone deacetylase inhibitor having theformula:

wherein: X is chosen from H and CH₃; Y is (CH₂)_(n) wherein n is 0-2; Zis chosen from (CH₂)_(m) wherein m is 0-3 and (CH)₂; A is a branchedaliphatic group having from 3 to 14 carbons; B is o-aminophenyl orhydroxyl group; and Q is a halogen, hydrogen, or methyl.
 2. Theinhibitor according to claim 1, wherein B is o-aminophenyl.
 3. Theinhibitor according to claim 1, wherein B is hydroxyl.
 4. A histonedeacetylase inhibitor having the formula:

wherein: X is chosen from H and CH₃; Y is (CH₂)_(n), wherein n is 0; Zis chosen from (CH₂)_(m) wherein in is 0-1 and (CH)₂; A is an α-branchedalkaryl group having 9 to 14 carbons, B is hydroxy, and Q is hydrogen.5. A histone deacetylase inhibitor chosen fromN-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;N-Hydroxy-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;N-(2-Amino-phenyl)-4-(2-propyl-pentanoylamino)-benzamide;N-Hydroxy-4-(2-propyl-pentanoylamino)-benzamide; 2-Propyl-pentanoic acid{4-[(2-amino-phenylcarbamoyl)-methyl]-phenyl]-amide; 2-Propyl-pentanoicacid (4-hydroxycarbamoyl-methyl-phenyl)-amide; 2-Propyl-pentanoic acid{4-[(2-amino-phenylcarbamoyl)-ethyl]-phenyl}-amide; 2-Propyl-pentanoicacid [4-(2-hydroxycarbamoyl-ethyl)-phenyl]-amide; 2-Propyl-pentanoicacid {4-[2-(2-amino-phenylcarbamoyl)-vinyl]-phenyl}-amide; and2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-vinyl)-phenyl]-amide. 6.A histone deacetylase inhibitor chosen fromN-(2-Amino-phenyl)-4-(phenylacetylamino-methyl)-benzamide;N-(2-Amino-phenyl)-4-[(4-phenyl-butyrylamino)-methyl]-benzamide;4-(Butyrylamino-methyl)-N-hydroxy-benzamide;N-hydroxy-4-(phenylacetylamino-methyl)-benzamide;N-hydroxy-4-[(4-phenyl-butyrylamino)-methyl]-benzamide;4-Butyrylamino-N-hydroxy-benzamide;N-hydroxy-4-phenylacetylamino-benzamide;N-hydroxy-4-(4-phenylbutyrylamino)-benzamide; andN-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-butyramide.
 7. A histonedeacetylase inhibitor chosen fromN-hydroxy-3-(4-phenylacetylamino-phenyl)-propionamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-4-phenyl-butyramide;N-(2-Amino-phenyl)-4-[(2-phenyl-butyrylamino)-methyl]-benzamide;N-(2-Amino-phenyl)-4-[(3-phenyl-butyrylamino)-methyl]-benzamide;N-hydroxy-4-(2-phenylbutyrylamino)-benzamide;N-hydroxy-4-(3-phenylbutyrylamino)-benzamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-phenyl-butyramide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-3-phenyl-butyramide;N-hydroxy-4-[(2-phenyl-butyrylamino)-methyl]-benzamide; andN-hydroxy-4-[(3-phenyl-butyrylamino)-methyl]-benzamide.
 8. A histonedeacetylase inhibitor chosen from 4-Benzoylamino-N-hydroxy-benzamide;4-(4-methyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-chloro)-Benzoylamino-N-hydroxy-benzamide;4-(4-bromo)-Benzoylamino-N-hydroxy-benzamide;4-(4-tert-butyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-phenyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-methoxyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-trifluoromethyl)-Benzoylamino-N-hydroxy-benzamide;4-(4-nitro)-Benzoylamino-N-hydroxy-benzamide; and Pyridine-2-carboxylicacid (4-hydroxycarbamoyl-phenyl)-amide.
 9. A histone deacetylaseinhibitor chosen fromN-hydroxy-4-(2-methyl-2-phenyl-propionylamino)-benzamide;N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;N-hydroxy-4-(3-phenyl-propionylamino)-benzamide;4-(2,2-Dimethyl-4-phenyl-butyrylamino)-N-hydroxy-benzamide;N-hydroxy-4-[methyl-(4-phenyl-butyryl)-amino]-benzamide;N-hydroxy-4-(2-phenyl-propionylamino)-benzamide;N-hydroxy-4-(2-methoxy-2-phenyl-acetylamino)-benzamide;4-Diphenylacetylamino-N-hydroxy-benzamide;N-hydroxy-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide; andN-(2-Amino-phenyl)-4-phenylacetylamino-benzamide.
 10. A histonedeacetylase inhibitor chosen fromN-(2-Amino-phenyl)-4-(5-phenyl-pentanoylamino)-benzamide;N-(2-Amino-phenyl)-4-(2-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(2,2-dimethyl-4-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-phenyl-propionylamino)-benzamide;N-(2-Amino-phenyl)-4-(4-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(3-methyl-2-phenyl-butyrylamino)-benzamide;N-(2-Amino-phenyl)-4-(2-methyl-2-phenyl-propionylamino)-benzamide;N-(2-Amino-phenyl)-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;and N-hydroxy-4-[2-(S)-phenylbutyrylamino]-benzamide.
 11. A histonedeacetylase inhibitor chosen fromN-hydroxy-4-[2-(R)-phenylbutyrylamino]-benzamide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(S)-phenyl-butyramide;N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(R)-phenyl-butyramide;N-hydroxy-4-(3-(S)-phenylbutyrylamino)-benzamide;N-hydroxy-4-(3-(R)-phenylbutyrylamino)-benzamide;N-hydroxy-4-[3-(S)-phenylbutyrylamino]-benzamide; andN-hydroxy-4-[3-(R)-phenylbutyrylamino]-benzamide.
 12. The inhibitoraccording to claim 1, wherein the inhibitor is an ester or salt.
 13. Apharmaceutical composition comprising the inhibitor according to claim1, and at least one pharmaceutically acceptable excipient.
 14. Theinhibitor according to claim 4, wherein m=0 and X=H.
 15. A histonedeacetylase inhibitor having the structure:

and pharmaceutically acceptable salts thereof.
 16. A histone deacetylaseinhibitor having the structure:

and pharmaceutically acceptable salts thereof.
 17. A histone deacetylaseinhibitor having the structure:

and pharmaceutically acceptable salts thereof.
 18. A compositioncomprising the inhibitor according to claim 15, wherein the compositionis enriched in the S-stereoisomer as compared to the R-stereoisomer. 19.The histone deacetylase inhibitor according to claim 4, wherein A is: